Outlining the skin-homing and circulating CLA+NK cells in patients with severe atopic dermatitis

Atopic dermatitis (AD) is a complex, multifactorial skin disease, characterized by pruritus and predominant Th2 inflammation. Innate immune cells may play a role in AD development and are composed of granulocytes, macrophages, innate-like T cells, and innate lymphoid cells. This study investigates the phenotypic and functional profile of circulating CLA+ natural killer (NK) cells and its role in the skin-homing to NK cells infiltrated in adults’ skin with AD. We selected 44 AD patients and 27 non-AD volunteers for the study. The results showed increased frequencies of both CLA+CD56bright and CLA+CD56dim NK cell populations in the peripheral blood, mainly in severe AD patients. Upon SEB stimulation, we observed an augmented percentage of CLA+CD56dim NK cells expressing CD107a, IFN-γ, IL-10, and TNF, reinforcing the role of staphylococcal enterotoxins in AD pathogenesis. Additionally, we demonstrated increased dermal expression of both NK cell markers NCAM-1/CD56 and pan-granzyme, corroborating the skin-homing, mostly in severe AD. Further studies are necessary to elucidate the potential role of NK cells in the chronification of the inflammatory process in AD skin, as well as their possible relationship with staphylococcal enterotoxins, and as practicable therapeutic targets.

reduction of mature CD56 dim CD57 + NK cells 2,5,10 .Notwithstanding, it has also been described small numbers of NK cells homing healthy skin, which indicates that further studies are necessary to elucidate the role of NK cells in AD, as well as how staphylococcal chronic colonization in AD skin influences this pathway 11 .
Cutaneous lymphocyte-associated antigen (CLA) is a glycoprotein with carbohydrate epitope expressed on the surface of different lymphocytes.CLA binds to E-selectin, expressed in endothelial cells and other tissues, during inflammation 12 .CLA + T cells participate in skin homing and represent the main T cell population in AD 12 and cutaneous lymphoma 13 lesions.NK cells express CLA at levels comparable to T cells, and IL-2 and IL-12 stimulation increases CLA expression in NK and T cells 13 .Functional studies focusing on CLA + T cells demonstrated that they are expressed in 15% of human circulating T cells, facilitating a differential migration of T cells to the skin, which indicates that they are functionally related to AD cutaneous inflammation 12 .In the dengue virus infection, peripheral blood NK cells showed CLA and other homing receptors (CCR5 and CXCR6) increased expression, in a pathway related to augmented plasmatic IL-18 levels, indicating an IL-18-dependent mechanism inducing NK cell proliferative response 14 .Our group has already demonstrated an increase in plasmatic IL-18 cytokine levels in AD patients 15 .However, the phenotypic and functional characteristics of CLA + NK cells in AD are unknown.
Previous studies from our group revealed decreased PBMC proliferation response to staphylococcal enterotoxin A (SEA) and other recall antigens and mitogens, as well as a reduction of the polyfunctional response of T cells to SEA, a mechanism associated with the inhibition of the transcription factor Early Growth Response 2 (EGR2) [15][16][17] , suggesting possible anergy of T lymphocytes.Although CD4 + T cells depletion in AD 16,17 , we hypothesized that an active NK cell-mediated innate response may occur, perhaps to overcome the T cell's anergic profile in response to staphylococcal enterotoxin B (SEB), which may reflect in AD cutaneous manifestations.In this study, we investigate the phenotypic and functional profile of circulating CLA + NK cells and their role in the crosstalk with NK cells infiltrated in adults' skin with AD.

Skin-homing CLA expression on NK cells subsets in adult AD
We assessed the expression of homing and activation molecules on the subtypes of NK cells in the peripheral blood of mild/moderate and severe AD patients.Figure 1a illustrates the NK cell analysis strategy, with our results showing no differences in the frequency of both CD56 bright CD16 − and CD56 dim CD16 + NK cell population comparing healthy controls (HC) and AD patients (Fig. 1b).On the other hand, a significant increase in the frequency of CLA expression was observed in subjects with severe AD, both in the CD56 bright (CD56 + CD16 − ) and CD56 dim (CD56 + CD16 + ) NK cell populations (Fig. 1c).This data suggests greater targeting of NK cells to the skin of individuals with severe AD.

NK cell response in adult AD induced by in vitro stimulation
In this step, we evaluated the functional profile of NK cells subsets regarding CD107a, IL-21R, IFN-γ, IL-10, and TNF production, after stimulation with a toll like receptor (TLR) 3 agonist (Poly I:C-viral stimuli), SEB (bacterial stimuli), and PMA/Ionomycin (positive control).Supplementary Figure S1 illustrates the gating strategy for NK cells.We observed an increased expression of IL-21R in the CLA + NK CD56 bright cell population at present in the baseline condition, which remained increased upon stimuli in severe AD patients in comparison to HC and mild/moderate AD groups (Fig. 2b).We did not detect differences in CD107a, IFN-γ, IL-10, and TNF in CLA + NK CD56 bright cells (Fig. 2a, c, d, e).As for CLA + NK CD56 dim cells, we observed that stimulation with SEB was the most significant method for inducing expression of CD107a, IFN-γ and IL-10 in severe AD patients (Fig. 2f, h, i).Similarly, SEB stimulated TNF expression in mild/moderate AD patients (Fig. 2j).The Poly I:C stimulation also increased the expression of CD107a and IL-21R in CLA + NK CD56 dim cells, in patients with severe AD (Fig. 2f, g).
Noteworthy, in order to proper stimulate NK cells cytokine production, PMA/Iono was adopted as a positive control, which corroborates with previous studies in the literature 18,19 .

NK expression profile in skin lesions from adults with AD
Considering the CLA + CD56 cells expression in the blood, we evaluate the NK cells migration to the skin through the expression of pan-granzyme and NCAM-1/CD56 (neuronal cell adhesion molecule-1), in AD skin compared to HC. Figure 3a shows the dermal expression profile from HC, mild/moderate AD, and severe AD individuals.We found an augmented expression of CD56 in both AD groups, and an increased expression of pan-granzyme in severe AD individuals compared to HC (Fig. 3b, c).We also evaluated NK cells assessing double-labeled cells expressing pan-granzyme and CD56 by immunofluorescence in skin sections of AD and HC samples.Figure 4 shows an increased expression of CD56 + cells co-expressing pan-granzyme at the dermis of severe AD patients.

Discussion
Our study demonstrated an increased ex vivo expression of both CLA + CD56 bright and CD56 dim NK cell population, mainly in severe AD patients, and an augmented functional ability to express IFN-γ, IL-10, and TNF under SEB stimulation in CLA + CD56 dim NK cells, reinforcing the role of staphylococcal enterotoxins in the AD pathogenesis.Additionally, we demonstrated an increased dermal expression of both NK cell markers NCAM-1/ CD56 and pan-granzyme, corroborating the skin-homing mostly in severe AD.
When evaluating NK cells in CLA + dependency, we noted an increased expression of NK CD56 bright and CD56 dim in severe AD.In fact, it has been described as an additive effect when CD56 dim NK cells were stimulated in vitro with a combination of IL-2 and IL-21 in peripheral blood from healthy donors 20 .In contrast, a study assessing patients with AD demonstrated that they have not only a global reduction in their blood NK cells but also a shift in subpopulations of NK cells, which is reflected by a loss of mature CD56 dim effector cells 5 .Other study revealed decreased frequencies of total CD56 + NK cells in AD supporting an increased NK cell apoptosis in the blood and extravasation in the inflamed skin 21 .In accordance with Mack et al. 5 , we believe that NK cells from AD patients may be undertaking an activation-induced cell death.More studies are needed to better elucidate the role of NK cells in the innate immune response of patients with AD 11 .
In the functional evaluation of NK cells, our study revealed increased expression of CD107a, IFN-γ, IL-10, and TNF under SEB stimulation in CLA + NK dim cells, reinforcing the role of staphylococcal enterotoxins in AD pathogenesis.Several studies have demonstrated that NK cell activation by staphylococcal enterotoxins increases IFN-γ 22,23 , granzyme, and perforin expression by NK cells, in a monocyte or T cell-dependent manner 24 .Poly I:C NK activation, viral ssRNA stimuli, elevated the expression of CD107a and IL-21R by CLA + NK dim cells in patients with severe AD, suggesting that antiviral cytotoxic response, mediated by TLR3 activation, is preserved in these patients 25 .
The increase in TNF expression at baseline in CD56 bright cells has already been evidenced in some inflammatory skin diseases (lichen planus, psoriasis, Sezary's syndrome, and mycosis fungoides), as well as in our findings in adults with AD 26,27 .However, we did not observe similar effect in CLA + NK CD56 bright cell population.We detected an increased expression of IL-21R in the CLA + NK CD56 bright cell population in the baseline condition, which remained increased upon stimuli in severe AD patients in comparison to HC and mild/moderate AD groups.It has been described that IL-21 exert effects on the expression of NK cell receptors, as well as increasing the expression of effector molecules, inducing perforin and granzyme B expression, and consequently increasing NK cell cytotoxicity in vitro 20,28 .www.nature.com/scientificreports/It is worth to mention that our study aimed to evaluate the functional response of CLA + NK cell subsets, which are not abundant in peripheral blood, but they still represent a relevant population for the understanding of AD pathogenesis.
To corroborate the NK cells skin-homing in AD lesions, we demonstrated that severe AD exhibited increased expression of pan-granzyme and NCAM-1/CD56 NK cell markers.Additionally, we found the expression of both NK cell markers in severe AD dermal specimens, in accordance with our previous findings.Previous studies described that granzyme-expressing cells are increased in lesions of patients with inflammatory skin diseases, such as AD, psoriasis, allergic contact dermatitis, and pityriasis rosea, compared with healthy skin, as well as plasma granzyme B levels being higher in patients with AD and psoriasis than in HC, with a positive correlation with Visual Analogue Scale (VAS) score.Our findings indicate that granzyme in severe AD cutaneous lesions, from NK cells, may exert a critical role in the initiation and perpetuation of itch and inflammation in AD, through the modulation of the innate response despite the adaptive response exhaustion 29 .
The potential role of NK cells in maintaining the inflammatory process in AD skin, as well as their possible relationship with staphylococcal enterotoxins and as practicable therapeutic targets, requires further studies.

Subjects
We enrolled 44 patients with AD (aged between 18 and 43 years; mean age: 28.93 ± 9.16; 24 males and 20 females) and 27 healthy non-AD volunteers (aged between 20 and 55 years; mean age: 36.48 ± 13.42; 8 males and 19 females) in this study.Thirteen AD patients and 12 healthy non-AD volunteers were selected for blood sample collection to be included in flow cytometry experiments, and 31 AD and 15 healthy non-AD volunteers skin samples were provided by our skin biorepository for immunohistochemistry experiments.AD was diagnosed according to the Hanifin & Rajka criteria 30 , and the disease severity was evaluated by the Eczema Area and www.nature.com/scientificreports/Severity Index (EASI) 31 .AD patients were classified as mild/moderate (n = 17), or severe (n = 27), according to EASI 31 .None of the patients were under systemic treatment with oral steroids or immunosuppressants.The study was approved by the Ethics Committee of the University of Sao Paulo School of Medicine, and informed consent was obtained from all subjects.All methods were performed in accordance with the relevant guidelines and regulations of this institution.Table 1 summarizes the demographic data of the study.

Immunohistochemistry of NK cells
Immunohistochemistry was performed on slices with a 4 µm thickness of paraffin-embedded samples, as already described [33][34][35] .The primary antibody Pan-granzyme (isotype Goat polyclonal IgG, SC-1969; Santa Cruz Biotechnology, Dallas, TX, USA) was utilized at 1:50 dilution, and the detection system was LSAB + System-HRP (K0690; DakoCytomation, Carpinteria, CA, USA); NCAM-1/CD56 (isotype Mouse monoclonal IgG1, ab200698; Abcam, Cambridge, MA, USA) was utilized at 1:100 dilution, and the detection system was Novolink Max Polymer Detection System (RE7280-K; Leica Biosystems, Newcastle Upon Tine, UK), and the chromogen used was DAB (3,3′ diaminobenzidine, D5637, Sigma).We performed scanning of immunohistochemically stained specimens using Aperio Scan-scope Cs (Aperio Technologies, Vista, CA, USA).Images of the scanned stained specimens were analyzed utilizing the software Image-Pro Plus version 4.5.0.29 (Media Cybernetics Inc., Bethesda, Maryland, USA) 34,36 .Using the software, we obtained the calculation of the total tissue distribution of pan-granzyme and NCAM-1/CD56 dividing the total number of doubly stained cells by the total area measured in the dermis.The expression of the measured markers was normalized by the total area and expressed in cells/μm 2 .

Statistical analysis
We analyzed the comparison of 2 groups using the non-parametric Mann-Whitney test and the comparison of 3 groups using the Kruskal-Wallis test, with Dunn's post-test (statistically significant when p < 0.05).

Figure 3 .
Figure 3. NK expression profile in skin lesions from adults with AD.Expression of NCAM-1/CD56 and granzyme in skin specimens from adults with AD assessed by immunohistochemistry. (a) Image of a skin specimen from a HC: NCAM-1/CD56 and pan-granzyme expression.NCAM-1/CD56 (b) and pan-granzyme (c) expression levels (cell/μm 2 ) in the dermis of the control group (HC, n = 15) compared with mild/moderate AD (n = 10) and severe AD (n = 21) patients, evaluated by immunohistochemistry. Lines represent medians with interquartile ranges of NK markers in skin specimens.**p < 0.01 and ****p < 0.0001.

Figure 4 .
Figure 4. Immunofluorescence staining of NK cell markers in AD skin.Double-label immunofluorescence panels showing that dermal cells express both NCAM-1/CD56 (green) and granzyme NK cells (red).Yellow arrows show the merge of NCAM-1/CD56 and granzyme cells expressing NK cells.